The mechanism of retroviral entry into cells involves binding of viral envelope (ENV) proteins to cellular receptors followed by a set of conformational changes in ENV giving rise to virus-cell membrane fusion. However, these events are not well understood. This proposal is aimed at better understanding this critical first step of viral replication using the avian leukosis virus (ALV)-receptor model system. The first specific aim is to determine the molecular basis for the existence of two functionally-distinct forms of the TVBS1 receptor for subgroups B, D, and E ALV. These studies will provide crucial new insights into the mechanism of entry used by these viruses and into the non-reciprocal receptor-interference pattern observed between these viruses. Furthermore, the results of these studies should also have important implications for understanding the biological significance of non-reciprocal receptor interference seen with other types of retroviruses. The second specific aim is to characterize conformational changes in ALV ENV glycoproteins following receptor-binding and low pH activation, and to test the hypothesis that these events are sufficient to stimulate ALV ENV-driven membrane fusion. These studies build upon our novel and unexpected finding that ALV ENV-driven membrane fusion requires receptor-binding followed by a low pH-activated step and will test the sufficiency of these events for viral entry. The third specific aim is to identify viral entry determinants of ALV ENV and receptor proteins, studies that will help us to build rational models for ALV entry into cells and that will be invaluable for validating the biological significance of crystal structures of ALV ENV-receptor complexes once they become available. The fourth specific aim addresses whether ALV use a clathrin-dependent endocytic pathway for entry into cells. Together, these studies will significantly advance our understanding of how retroviruses enter cells. Understanding this process in detail should lead to better antiviral therapeutic strategies and aid with viral targeting approaches for gene therapy protocols.